An 82-kD protein has been purified from the undercoat of the adherens junction isolated from the rat liver. The purification scheme includes low salt extraction followed by DEAE-cellulose ion exchange, DNase I- actin affinity, and carboxyl methyl-cellulose ion exchange chromatographies. The purified 82-kD protein was essentially free of contaminants as judged by SDS-PAGE combined with silver staining. The substoichiometric 82-kD protein largely inhibited the actin filament assembly; when the molar ratio of the 82-kD protein to G-actin was 1:1,000, the viscosity was reduced to 28% of the control value. Direct electron microscopic studies revealed that the 82-kD protein selectively inhibited monomer addition at the barbed ends of actin filaments. By use of the antibody raised against the 82-kD protein, this protein was shown by immunofluorescence microscopy to be localized at the cell-to-cell adherens junction in various types of cells. In contrast, the 82-kD protein was not concentrated at the cell-to- substrate adherens junctions (focal contacts). These findings have led us to conclude that the 82-kD protein is a barbed end-capping protein which is associated with the undercoat of the cell-to-cell adherens junction. Hence, we have tentatively designated the 82-kD protein as radixin (from the Latin word radix meaning root).

Abstract. Hemidesmosomes (HDs) mediate cell adhesion to the extracellular matrix and have morphological association with intermediate-sized filaments (IFs) through cytoplasmic plaques . Though several proteins have been located in HDs, most of them have not been well characterized, with the exception of the 230-kD antigen of bullous pemphigoid (BP), an autoimmune skin blistering disease. Only recently we have succeeded in isolating HDs from bovine corneal epithelial cells and in identifying five major components on SDS-PAGE (Owaribe K., Y. Nishizawa, and W. W. Franke . 1991 . Exp. Cell Res . 192 :622-630) . In this study we report on immunological characterization of one of the major components, termed HDl, with an H EMIDESMOSOMES (HDs)' and focal adhesions are cell junctions that mediate adhesion to the extracellular matrix (ECM) . These two plasma membrane junction types have common ultrastructural features ; e.g., both are associated with cytoskeeeool elements through electron-dense cytoplasmic plaques. They thus provide us with models to explore the mechanisms of transmembrane connection between the ECM and the cytoskeleton. In focal adhesions associated with actin-containing microfilaments (for reviews see references 2 and 50), a number ofproteins are known to be involved, including ECM receptors of the integrin family (5,12,17,33) and plaque components such as talin (1) and vinculin (29) . Yet our knowledge about the molecular architecture of the ECM-to-microfilaments junctions is still incomplete.The HD, primarily found in the basement membrane zone (BMZ) of stratified and complex epithelia, has a complex structure: a cytoplasmic plaque associated with intermediate filaments (IFs), a subbasal dense plate with anchoring filaments in the basal lamina, and anchoring fibrils in collagenous connective tissues (6,16,39). The morphological appearance is essentially that of half of the desmosome of intercellular junctions. However, although several des- apparent molecular mass of 500 kD. Immunofluorescence microscopy showed colocalization of HDl with BP antigen at the basement membrane zone of those tissues that have typical HDs, including skin epidermis, corneal and tracheal epithelia, and myoepithelium . In cultured keratinocytes, HDl demonstrated colocalization with BP antigen in the precise way, while being absent from focal adhesions . Immunoelectron microscopy revealed that an epitope of HDl was located on the cytoplasmic side of HDs . Taking all these results together, we conclude that HDl is a new hemidesmosomal component . Interestingly, HDl also exists in endothelial and glial cells, which lack typical HDs . mosomal proteins have been identified (e.g., references 4, 26; for review see reference 35) since the establishment of appropriate isolation procedures (e.g., reference 36), none of them have been found in HDs (13, 35) . Thus, in marked contrast to other celljunctions, little information is available about the molecular components of HDs, with the exception ofthe well-characterized 230-kD antigen r...

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

Here, we investigated the expression of the claudin family of tight junction transmembrane proteins in the developing mouse submandibular gland. Data obtained by reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence microscopy showed the expression and localization of claudin-3 to -8, -10, and -11 at epithelial tight junctions. Examination of the glands taken from embryonic day (E) 14, E16, and newborn mice revealed differential expression patterns of these claudins in the developing epithelium. Claudin-3, -5, and -7 were expressed in all of the luminal epithelial cells of the ducts at all of the developmental stages examined and in those of terminal tubules at E16 and later. Claudin-4 was expressed mainly in the ducts at all the developmental stages. The expression of claudin-6 and -8 was also restricted to the ducts at E14 and E16; but after birth, the former was undetectable, whereas the latter was expressed in both the ducts and terminal tubules. Claudin-10 and -11 were detectable mainly in the terminal tubules at E16 and later. In addition to being found in the epithelium, claudin-5 was also expressed in certain mesenchymal cells, probably endothelial cells. These results will provide a valuable resource for further investigation of tubulogenesis and physiological regulation of claudin-based tight junctions. Developmental Dynamics 231:425-431, 2004.

In the present study, the role of a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), in organ development was investigated by using developing mouse submandibular gland (SMG), in which the EGF receptor signaling and heparan sulfate chains have been implicated. HB-EGF mRNA was detected in developing SMG by RT-PCR analysis and was expressed mainly in epithelium and weakly in mesenchyme of the embryonic SMG. Epithelial morphogenesis was inhibited by a synthetic peptide corresponding to the heparin-binding domain of HB-EGF and by anti-HB-EGF neutralizing antibody. An in vitro assay using an EGF receptor ligand-dependent cell line, EP170.7 cells, allowed us to detect the growth factor activity in SMG-conditioned media, which was significantly reduced by anti-HB-EGF antibody. Furthermore, treatment of SMG rudiments with the hydroxamate-based metalloproteinase inhibitor OSU8-1, which inhibits processing of EGFR ligands including HB-EGF, markedly diminished the growth factor activity in conditioned media and resulted in almost complete inhibition of SMG morphogenesis. The inhibitory effects on morphogenesis were reversed, though partially, by adding the soluble form of HB-EGF. Our results provide the first evidence that HB-EGF is a crucial regulator of epithelial morphogenesis during organ development, highlighting the importance of its processing by metalloproteinases.